A series of products and by-products of naturally occurring metabolic processes in microorganisms, animal cells and plant cells has utility for a wide array of industries, including the feed, food, cosmetics and pharmaceutical industries. These molecules, which are collectively termed “fine chemicals”, also include, for example, lipids and fatty acids, one representative class of which are the polyunsaturated fatty acids. Fatty acids and triglycerides have a multiplicity of uses in the food industry, in animal nutrition, in cosmetics and in the pharmacological sector. Depending on whether they take the form of free saturated or unsaturated fatty acids or else triglycerides with an increased content of saturated or unsaturated fatty acids, they are suitable for a variety of uses; thus, for example, polyunsaturated fatty acids (PUFAs) are added to baby formula for increasing the nutritional value. Moreover, PUFAs have a positive effect on the cholesterol level in the blood of humans and are therefore useful for protection against heart disease. Thus, they are used in a variety of dietetic foods or in medicaments.
Microorganisms which are particularly useful for the production of PUFAs are microorganisms such as Thraustochytria or Schizochytria strains, algae such as Phaeodactylum tricornutum or Crypthecodinium species, ciliates such as Stylonychia or Colpidium, fungi such as Mortierella, Entomophthora or Mucor. Through strain selection, a number of mutant strains of the respective microorganisms have been developed which produce an array of desirable compounds including PUFAs. However, the selection of strains in which the production of a particular molecule is improved is a time-consuming and difficult process.
Alternatively, fine chemicals can conveniently be produced via producing, on a large scale, plants which have been developed in such a way that they produce the abovementioned PUFAs. Particularly well suited plants for this purpose are oil crop plants which comprise large amounts of lipid compounds, such as oilseed rape, canola, linseed, soybean, sunflower, borage and evening primrose. However, other useful plants comprising oils or lipids and fatty acids are also well suited as mentioned in the detailed description of this invention. By means of conventional breeding, an array of mutant plants has been developed which produce a spectrum of desirable lipids and fatty acids, cofactors and enzymes. However, the selection of novel plant cultivars in which the production of a particular molecule is improved is a time-consuming and difficult process or indeed impossible if the compound does not occur naturally in the respective plant, as is the case of polyunsaturated C18-, C20-fatty acids and C22-fatty acids and those with longer carbon chains.
Owing to the positive properties of unsaturated fatty acids, there has been no lack of attempts in the past to make available genes which are involved in the synthesis of fatty acids or triglycerides for the production, in various organisms, of oils with a modified content of unsaturated fatty acids. Thus, WO 91/13972 and its US equivalent describe a Δ9-desaturase. A Δ15-desaturase is claimed in WO 93/11245 and a Δ12-desaturase is claimed in WO 94/11516. Δ6-desaturases are described in WO 93/06712, U.S. Pat. No. 5,614,393, WO 96/21022 and WO 99/27111. Further desaturases are described, for example, in EP-A-0 550 162, WO 94/18337, WO 97/30582, WO 97/21340, WO 95/18222, EP-A-0 794 250, Stukey et al., J. Biol. Chem., 265, 1990: 20144-20149, Wada et al., Nature 347, 1990: 200-203 or Huang et al., Lipids 34, 1999: 649-659. A Δ6-palmitoyl ACP desaturase is described and claimed in WO 96/13591. However, the biochemical characterization of the various desaturases is incomplete as yet since the enzymes, being membrane-bound proteins, can only be isolated and characterized with great difficulty (McKeon et al., Methods in Enzymol. 71, 1981: 12141-12147, Wang et al., Plant Physiol. Biochem., 26, 1988: 777-792).
In yeasts, both a shift in the fatty acid spectrum toward unsaturated fatty acids and an increase in productivity have been found (see Huang et al., Lipids 34, 1999: 649-659, Napier et al., Biochem. J., Vol. 330, 1998: 611-614). However, the expression of the various desaturases in transgenic plants did not show the desired success. While a shift in the fatty acid spectrum toward unsaturated fatty acids was demonstrated, it emerged that the synthesis performance of the transgenic plants dropped drastically, i.e. only smaller amounts of oils were isolated compared with the original plants.
Neither yeasts nor plants naturally produce polyunsaturated C20- and/or C22-fatty acids with at least two double bonds in the fatty acid molecule, such as arachidonic acid (ARA) and/or eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA).
This is why there still exists a great demand for novel genes which encode enzymes which are involved in the biosynthesis of unsaturated fatty acids and which make possible their production on an industrial scale. None of the prior-art biotechnological methods for the production of polyunsaturated fatty acids yields the abovementioned fatty acids in economically useful quantities.
It is an object of the present invention to provide further enzymes for the synthesis of polyunsaturated fatty acids and to use these enzymes, with or without other enzymes, in a method for the production of polyunsaturated fatty acids.